1. Field of the Invention
The present invention relates in general to plant genetic engineering, and in particular to novel 5' and 3' regulatory sequences, including a novel intron contained within the leader of the 5' regulatory sequence, useful in the expression of genes in plants during early seed development and throughout seed development. These regulatory sequences can be operably linked to DNAs encoding selected protein products, and the combinations can be incorporated into recombinant vectors to facilitate the expression of these DNAs in transgenic plants.
2. Description of Related Art
The expression of a plant gene existing in the form of double stranded DNA involves transcription of messenger RNA (mRNA) from one strand of the DNA by RNA polymerase and subsequent processing of the MRNA primary transcript inside the nucleus. This processing involves a 3' non-coding region which adds polyadenylate nucleotides to the 3' end of the MRNA.
Transcription of DNA into mRNA is regulated by a region of DNA usually referred to as the promoter. The promoter region contains a sequence of bases that signals RNA polymerase to associate with the DNA and initiate the transcription of mRNA using one of the DNA strands as a template to make a corresponding complementary strand of RNA.
Recent advances in genetic engineering have provided the requisite tools to transform plants to contain and express foreign genes (Kahl et al. (1995) World Journal of Microbiology and Biotechnology 11: 449-460). It is now possible to produce plants that have unique characteristics of agronomic and crop processing importance. The ability to select the tissues in which to express such foreign genes and the time during plant growth in which to obtain expression of such foreign genes is possible through the choice of appropriate promoters that turn on these genes. A wide range of promoters is known for various plants, plant tissues, and developmental stages (McKinnon et al. (1995) Journal of Cereal Science 22: 203-210; Baerson et al. (1993) Plant Molecular Biology 22: 255-267; Domanskii et al. (1993) Biopolimery Kletka 9:3-18).
The soybean is an important plant for genetic engineering as it can be transformed to express foreign genes (Delannay et al. (1995) Crop Science 35: 1461-1467), and because it possesses valuable characteristics that can be improved by introducing certain genes.
Promoters useful in expressing foreign genes in soybean seeds and other seeds/cotyledons are known. For example, promoters from genes encoding seed storage proteins of various dicotyledonous plants have been identified. The major seed storage protein in soybean is .beta.-conglycinin. The promoters from both the .alpha.' and .beta. subunits of .beta.-conglycinin have been identified (Slighton et al. (1987) Planta 172: 356-363; Barker et al. (1988) Proceedings of the National Academy of Sciences, USA 85: 458-462). Promoters from genes encoding seed storage proteins of other species are also known. Two 2S albumin promoters of Arabidopsis thaliana have been identified (Conceicao et al. (1994) The Plant Journal 5(4): 493-505), the promoter of a 2S albumin gene of Brassica napus has been identified (Mats et al. (1991) European Journal of Biochemistry 197: 741-746), and a promoter from the .beta.-phaseolin gene of Phaseolus vulgaris has been identified (Bustos et al. (1988) Plant Cell 1: 839-853). Since seed storage proteins accumulate to high levels in seeds, the promoters from these genes have the potential to drive the expression of target genes to high levels in seeds of transgenic plants.
In addition to the promoter, the 5' untranslated regions of genes, introns from genes, and the 3' non-coding regions of genes have also been observed to play significant roles in the regulation and enhancement of gene expression. For example, 5' untranslated leaders have been observed to increase transient expression of heterologous genes (Hobbs et al. (1990) Plant Cell Reports 9: 17-20). Introns have also been observed to increase transient expression of heterologous genes (Callis et al. (1987) Genes and Development 1: 1183-1200). In addition to enhancing gene expression, introns that function in the regulation of gene expression have also been identified. For example, the GapAl promoter from maize requires the first intron of the GapA1 gene as well as surrounding exon border sequences in order to express in cultured cells (Donath et al. (1995) Plant Molecular Biology 28: 667-676). 3' noncoding regions of genes have also been observed to be involved in the regulation of gene expression. For example, enhancers contained within the 3' noncoding regions of genes have been required for the activation of tissue-specific expression in promoter-GUS fusions (Dietrich et al. (1992) The Plant Cell 4: 1371-1382; Larkin et al. (1993) The Plant Cell 5: 1739-1748).
The use of promoters from seed storage protein genes to drive the expression of heterologous genes in transformed plants is limiting in that storage proteins are predominantly expressed late in seed development, i.e., only during the midmaturation stages of development. Thus, there is a need for strong promoters that facilitate expression of genes during early seed development. Promoters active during early seed development possess the advantage of causing the selective accumulation of desirable gene products in seeds during early seed development, facilitating the enhanced production and accumulation of such desirable gene products in seeds, i.e., plant tissues that can be conveniently and economically harvested and processed.
While present technology permits the transformation of plants with genes encoding selected products, the expression of such genes is either ubiquitous if the promoter is constitutive, or is regulated in a temporal or tissue-dependent manner if the promoter is stage- or tissue-specific. Continuous expression precludes production at particular stages or in specific tissues, and can adversely affect yield due to increased energy demands associated with prolonged synthesis of the product. Tissue- or stage-specific expression permits greater control over the temporal and spatial accumulation of desired products. Thus, promoter sequences that control the expression of desired genes in a tissue-specific, stage-specific manner that can be employed in recombinant constructs for the transformation of plants, and that would facilitate greater control over the location and timing of expression of introduced genes and reduce the possibility of deleterious effects on overall plant growth, are highly desirable.